Method for determination of water treatment polymers

ABSTRACT

A method for determining the presence and/or concentration of a water treatment polymer in an aqueous sample, comprising producing a polyclonal or monoclonal antibody to the water treatment polymer;, and using the antibody so produced as a reagent in an immunoassay, conducted on the aqueous sample.

[0001] The present invention relates to a determination method, inparticular to a method, based on immunoassay, for the determination ofwater treatment chemicals in aqueous media, and to novel antibodies andhybridomas useful in the new method.

[0002] The majority of natural waters, and aqueous systems in general,contain dissolved salts of metals such as calcium, magnesium, barium andstrontium. When the natural water or aqueous system is heated, thedissolved salts may be converted to insoluble salts, and thereupondeposited as scale on any heat transfer surfaces in contact with thewater or aqueous system. Insoluble salt scale may be formed even whenthe water or aqueous system is merely concentrated, without beingheated.

[0003] Such precipitation and scale deposition are troublesome and canresult in an increase in the costs required to maintain aqueous systemsin good working order. Among the problems caused by scale deposits areobstruction of fluid flow, impedance of heat transfer, wear of metalparts, shortening of equipment life, localised corrosion attack, poorcorrosion inhibitor performance and unscheduled equipment shutdown.These problems can arise, e.g. in any circulating water system such asthose used in oil drilling wells, steam power plants, water desalinationplants, reverse osmosis equipment, heat exchange equipment and equipmentconcerned with the transport of products and by-products in aqueousmedia, e.g. fly-ash formed during the combustion of coal, in theproduction of electricity.

[0004] A number of additives, notably polycarboxylates, have beenprovided as effective scale inhibitors for addition to aqueous systems.

[0005] Likewise, natural waters and aqueous systems are corrosivetowards metals which are in operational contact with them. Consequently,such aqueous systems must be treated with a corrosion inhibitor, e.g. aphosphonate, in order to prevent deterioration of such metals, e.g.pipelines.

[0006] Although water treatment chemicals can be effective at very lowconcentrations, a certain minimum concentration must be maintained ifthe aqueous system is to operate trouble-free. With the passage of time,loss of the water treatment chemical from the system occurs andreplenishment is necessary to avoid the above-mentioned operationalproblems. On the other hand, use of excess of water treatment chemicalincreases operational costs. The need to balance treatment, chemicaleffectiveness and cost has led, therefore, to the development of methodsand devices for monitoring the level of water treatment chemicals inaqueous systems.

[0007] For example, colourimetric methods are available for thedetermination of scale inhibitors, e.g. polycarboxylates. Colorimetricmethods, however, have the disadvantage that they are subject tointerference from extraneous materials. In oil field applications, forinstance, interference arises mainly from iron and oil-derived organicmaterials.

[0008] In an attempt to overcome this interference problem, asample-preparation (pretreatment) cartridge maybe employed, in whichinterfering species are removed and the water treatment chemical isconcentrated. Unfortunately, however, such techniques can result in lossof the water treatment chemical being determined due to competition fromthe organics for adsorption sites on the cartridge. Such methods aretime consuming, lack robustness and the required sensitivity (limits ofdetection only 1-2 ppm). In addition they require a certain amount ofexpertise in order to be used effectively to conduct the requireddetermination.

[0009] More recently, immunological methods have been developed for thedetermination of organic compounds.

[0010] Immunological methods for determining proteins, cells, hormones,vitamins, drugs and mycotoxins etc. have been known for many years, andhave been widely reported in the literature. In such methods, an animal,often a mouse or rabbit, is immunize, either with an analyte or aprotein-analyte conjugate. The antibodies produced by the animal arethen used, in the form of an immunoassay, to determine the analyte.These methods are based upon the specific reaction between the analyteand the antibody.

[0011] The immunoassays which have been reported in the literatureincorporate antibodies that have been raised to natural molecules.Recently, however, EP 260829A, has disclosed novel mono- and polyclonalantibodies which are reactive with chlorinated phenols, especiallypentachlorophenol. The antibodies can then be used to identify and assaypentachlorophenol, which is widely used as a pesticide and preservative.

[0012] We have now succeeded in applying an immunoassay method to thedetection of water treatment polymers in aqueous solution, to provide adetermination method which is sensitive, specific, rapid, robust andwhich can be operated by relatively inexperienced personnel—this has notbeen achieved by such methodology before the present application.

[0013] It is surprising that an antibody can be raised effectively tomolecules which are polydisperse i.e. having differing molecular weightswhich vary considerably in size and shape. The competitive assay resultsdemonstrate that the antibodies are raised to the core active centre ofthe molecules i.e. a moiety which is present in every molecule in theproduct although the number of repeating monomer units can vary.

[0014] Accordingly, the present invention provides a method fordetermining the presence and/or concentration of a water treatmentpolymer in an aqueous sample, comprising the production of polyclonal ormonoclonal antibody to the water treatment polymer, and using theantibody so produced as a reagent in an immunoassay conducted on theaqueous sample.

[0015] The present invention also provides a method for determining thepresence and/or concentration of a water treatment polymer in an aqueoussample, comprising an effective amount of a monoclonal antibody orpolyclonal antibody which has been raised to the water treatmentpolymer, in association with an acceptable carrier.

[0016] Preferred water treatment polymers, for determination in theprocess of the present invention, are phosphorus acid containingcarboxylic acid telomers having the formula I:

[0017] or salts thereof, in which R″ is hydrogen, methyl or ethyl, R ishydrogen, C₁-C₁₈ alkyl, C₅-C₁₂ cycloalkyl, aryl, aralkyl, a residue offormula:

[0018] in which R″ has its previous significance and the sum of m and nis an integer of at most 100, or R is a residue —OX in which X ishydrogen or C₁-C₄ alkyl, and R¹ is a residue —OX in which X has itsprevious significance.

[0019] The telomers of formula I, and their production are described inmore detail in U.S. Pat. No. 4,046,707.

[0020] Particularly preferred telomers of formula I are those having theformula IA:

[0021] in which the sum of m′ and n′ is an integer ranging from 4 to 32,especially, 15 to 20.

[0022] Other preferred water treatment polymers, for determination inthe process of the present invention are hydrolyzed terpolymers ofmaleic anhydride with other monomers the molar ratio of maleic anhydrideto the other monomers ranging from 2.5:1 to 100:1 and the molecularweight of the terpolymer being below 1000. Such terpolymers aredescribed in U.S. Pat. No. 4,126,549.

[0023] Preferred ratios of monomers in the terpolymer are in the rangeof 2½-3½:1 of maleic anhydride to other monomers. Preferred othermonomers are vinyl acetate acid and ethyl acrylate.

[0024] These ratios are those used in the preparation of the cotelomerof formula II and are not necessarily the ratios to be found in thefinal cotelomer.

[0025] Other examples of preferred water treatment molecules includeother polyacrylic acid polymers; copolymers of acrylic acid andacrylamidomethylpropane sulphonic acid (AMPS); copolymers of acrylicacid and vinyl acetate; polymaleic acid; hydrolysed polymaleic acid;terpolymers of maleic acid, ethyl acrylate and vinyl acetate; copolymersof acrylic acid and maleic anhydride; copolymers of maleic acid andsodium allyl sulphonate; and copolymers of maleic anhydride andsulphonated styrene-and vinyl sulphonic acid telomers.

[0026] With respect to aqueous systems in which water treatment polymersto be determined may be present, of particular interest are the aqueoussystems employed in cooling water plant steam generating plant,sea-water evaporators, reverse osmosis equipment, paper manufacturingequipment, sugar evaporator equipment, soil irrigation plant,hydrostatic cookers, gas scrubbing systems, closed circuit heatingsystems, aqueous-based refrigeration systems and down-well systems.

[0027] The antibody used in the method and composition of the presentinvention may be produced by known techniques.

[0028] For the production of polyclonal antibodies which are reactivewith a particular water treatment polymer, firstly an immunogenicconjugate of the polymer and a macromolecule carrier may be produced; ananimal may then be immunized with the conjugate, the polymer alone,adjuvant or a discrete mixture of each; blood may be removed from theanimal and the serum separated from the blood; and finally thepolyclonal antibodies may be recovered from the serum.

[0029] It may be preferred, however, to use monoclonal antibodies, whichare reactive with specific epitopes on the water treatment polymer, inthe method and composition of the present invention, especially in viewof their superior specificity for a particular polymer. Monoclonalantibodies may be obtained by the technique first described by Kohlerand Milstein, Nature, 265:495 (1975). This technique comprises providingan immunogenic form of the specific water treatment polymer, immunizingan animal with such; obtaining antibody-producing cells from the animal;fusing the cells so obtained with myeloma cells to produce hybridomas;selecting from the hybridomas a hybridoma which produces an antibodywhich reacts with the specific water treatment polymer, and thenisolating the monoclonal antibody from the selected hybridoma.

[0030] Water treatment polymers generally have low molecular weights anddo not, per se, induce the production of antibodies. They can be used ashapten, however, in combination with a higher molecular weight,immunogenic carrier, such as a protein, using e.g. the techniquedisclosed by Albro et al. Toxicol Appl. Pharmacol 50,137-146 (1979).

[0031] The conjugate so obtained may then be used to immunize an animalhost, by conventional techniques, e.g. inoculation. The animal host maybe, e.g. a rabbit or a rodent such as a rat or mouse.

[0032] After the host animal has produced antibodies to the administeredconjugate, polyclonal antibodies may be recovered from the animal byconventional techniques.

[0033] For example, blood may be removed from the animal and serum maybe separated from the blood so removed. The desired antibodies may thenbe removed from the serum, e.g. by affinity purification or saltfractionation.

[0034] To produce monoclonal antibodies to the water treatment polymer,cells which produce antibodies may be recovered from the immunizedanimal. B lymphocytes removed from the animal's spleen are preferred.

[0035] The removed cells are fused with myeloma cells to producehybridomas, which are then separated, again using standard techniquessuch as cloning by limiting dilution.

[0036] Once the hybridomas have been separated a selection is made toascertain those which produce antibodies to the specific water treatmentpolymer to be determined in the method of the present invention. Therelevant specific hybridomas can then be isolated by known methods, andthe relevant antibodies secreted from them by conventional techniques.

[0037] The following examples further illustrate the present invention.

EXAMPLE 1

[0038] 1. Preparation of Protein Conjugates

[0039] A telomer (Telomer 1) derived from 16 moles of acrylic acid and 1mole of hypophosphorous acid and produced by the method of U.S. Pat. No.4,046,707 is bound to a carrier protein keyhole limpet haemocyanin (KLH)using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride(EDC). In addition the product is bound to a second protein, ovalbumin(OVA) for screening purposes.

[0040] Essentially 2 mg of KLH or OVA are dissolved with 200 μl ofdeionized water. In addition, 2 mg of the peptide to be coupled aredissolved in 0.5 ml of conjugation buffer (0.1M(2-(N-morpholino)-ethanesulfonic acid) MES, 0.9M sodium chloride NaCland 0.02% sodium azide NaN₃, pH 4.7).

[0041] The 500 μl of peptide solution are added to the 200 μl of carrierprotein solution. For OVA conjugation, this solution is added to 10 mgof EDC and dissolved by gentle mixing. For KLH conjugation, the 10 mg ofEDC are dissolved in 1 ml of deionized water and 50 μl of this solutionare added immediately to the carrier-peptide solution.

[0042] The reaction proceeds for 2 hours at room temperature. Anyprecipitate is removed using centrifugation prior to purification.

[0043] The conjugate is purified using gel filtration or Sephadex G50(0.5×5 cm). The column is washed using 5 ml of phosphate buffered salinePBS. The peptide carrier mixture is applied directly to the top of thecolumn and the eluate collected. 0.5 ml aliquots of PBS are added andeach fraction is collected in a separate tube. 15 mls of PBS are addedto elute both the conjugate and the peptide. The immunogen elutesbetween fractions 4-6, and the free peptide and reagents after fraction8.

[0044] The hapten-carrier ratios are determined spectrophotometicallyand by assessment of the concentrations of the reactants followingconjugation. The molar ratio of polymer per 100,000 mol. wt of carrieris 6-11.

[0045] 2. Immunisation of Animals

[0046] a) Mice (NZB/NZW F1 hybrid females and BALB-c females), 6-8 weeksold, receive 0.2 mg polymer in 0.1 ml 0.15M NaCl solution (saline) mixedwith 0.1 ml Freunds complete adjuvant (FCA) and 100 μg polymer conjugate(by protein concentration) in 0.1 ml saline. Thereafter animals areinjected every 18-21 days with the same antigen preparations and dosesexcept that Freunds incomplete adjuvant (FIA) is substituted for FCA.All injections are intraperitoneal and animals sacrificed for blood orspleens.

[0047] b) Rats (Sprague-Dawley females) aged 12-16 weeks are injectedwith the identical protocol indicated in (2a). Blood is obtained byheart puncture.

[0048] c) Rabbits (NZW-female) aged 4 months, are injected asfollows—day 0, intramuscular; day 14, intramuscular, day 24intraperitoneal. All treatments contain 50 ug protein or 200 ugpolymer/0.2 ml and are given in conjunction with 0.2 ml FCA (day 0), 0.2ml FIA (day 14), 0.2 ml saline (day 24). Blood is obtained on day 34 byvenepuncture, allowed to clot at room temperature and the serumseparated by centrifugation (2000 g, 15 min, 4° C).

[0049] 3. Monclonal Antibody Production

[0050] Mice, immunised as indicated above, are injected with polymer orconjugate (at the doses shown in 2a) 3 days prior to sacrifice.

[0051] The spleens are removed and the splenocytes isolated bydissection into Hanks Balanced Salt Solution. These spleen cells arefused with cells from the X63.Ag 8 6.5.3 murine myeloma line, inexponential growth, in a ratio of 4:1 by the addition of 1 ml 46% (w/v)polyethylene glycol 1550 (Serva) in RPMI 1640 with gentle mixing for 3min at 37° C. After standing for 2 min at room temperature, the mixtureis slowly diluted by the drop-wise addition of 20 ml RPMI 1640 over 5min, followed by standing at room temperature for 10 min. After washingtwice with RPMI 1640, the cells are incubated for 2 hr at 37° C. inbicarbonate-buffered RPMI 1640, supplemented with 10% (v/v) fetal calfserum, 2 mmol/l L-glutamine, 50 IU/ml penicillin and 50 ug/mlstreptomycin (Flow) and containing 1×10⁻⁴ mol/l hypoxanthine and1.6×10⁻⁵ mol/l thymidine (HT medium). The cell suspensions (100 ul) arethen dispensed into 96-well tissue culture plates (Costar) at threedifferent concentrations (2.5, 1.25 and 6×10⁶ cells/ml). Finally, 200 ulHT medium containing 4×10⁻⁷ mol/l aminopterin (HAT medium) are added toeach well. The plates are incubated at 37° C. in a humidified atmosphereof 5% CO₂ in air. Hybridoma cells are initially grown in HAT medium butthis is eliminated after 14 days by step-wise replacement with HTmedium. Supernatant liquids are screened for specific antibody byindirect non-competitive ELISA 14-18 d post-fusion. Specific hybridomasare subsequently expanded into flasks and cloned three times or until100% cloning efficiency is obtained. This procedure is carried out bylimiting dilutions in 96-well tissue culture plates containing a feederlayer of spleen cells (2×10⁵ cells/well) from non-immunized NZB/DALB-Chybrid mice. Cell lines of interest are maintain in vitro in culturemedium and are frozen, at a concentration of 5×10⁶ cells/ml, in RPMI1640 containing 30% bovine serum and 15% dimethyl sulphoxide (Sigma) andstored in liquid nitrogen (Islam, M. S. and Stimson, W. H. Lett. Appld.Microbiol., 4, 85-89 (1987).

[0052] 4. ELISA Procedures

[0053] a) Indirect non-competitive ELISA—for screening hybridomasupernatants and sera from animals for the presence of antipolymerantibodies.

[0054] i) Flat-bottomed 96-well microtire plates (Dynatech) are coatedwith polymer conjugate—10 ug protein/1 ml 0.02M Tris/HCl buffer, pH 9.0.Aliquots (100 ul per well) are distributed into microtitre plates andincubated for 1 h at 37° C. The solution is then removed and replacedwith 100 ul 1% (w/v) BSA solution in 0.02M Tris/HCl, pH 9.0, for 30 minat 37° C. Thereafter, the plates are washed (×4) with 0.2M Tris/HClbuffer pH 7.4 containing 0.2M NaCl and 0.05% (v/v) Tween 20 (washbuffer). These plates may be dried in vaccuo and stored dry for up toone year or used immediately for assays.

[0055] ii) Hybridoma supernatants or animal sera (dilutions 1:10 to1:10⁵ commonly) are added to the plates—100 ul per well. Followingincubation for 45 min, 37° C. the plates are washed ×3 with wash buffer.

[0056] iii) Sheep anti-mouse γ-globulins—horse radish peroxidaseconjugate (SAPU, Carluke, Scotland) is diluted 1:2000 in 0.15M NaClcontaining 25% (v/v) sheep serum. Aliquots (100 ul) are added to eachwell and incubated for 45 min at 37° C. before washing ×3 with washbuffer. Enzymic activity (A450) is measured with 200 ultetraethylbenzidine substrate, pH 5.5, the reaction is stopped ater 30min, room temperature with 50 ul 2M H₂SO₄.

[0057] b) Sandwich ELISA—for estimating polymer concentrations insamples.

[0058] i) Antisera are precipitated with (NH₄)₂SO₄ solution andredissolved in 0.15M NaCl solution to give a concentration of 15 mg/ml.This is diluted in 0.02M Tris/HCl, pH 9.0 from 1:500 to 10,000 and usedto coat microtitre plate wells (100 ul per well) for 1 h at 37° C. Theplate is washed ×5 with wash buffer before use.

[0059] ii) Polymer standards (10 ng/ml to 20 ug/ml) 0.15M NaCl solutionand samples (100 ul) are added to wells for 45 min at 37° C. The plateis washed ×3 in each buffer.

[0060] iii) Antibody/antiserum—enzyme conjugate preparation is achievedby periodate coupling of horse radish peroxidase (HRP).

[0061] 5 mg of HRP is resuspended in 1.2 ml of water. 0.3 ml of freshlyprepared 0.1M sodium periodate in 10 mM sodium phosphate (pH 7.0) isadded.

[0062] The solution is incubated at room temperature for 20 min prior todialysing the HRP solution versus 1 mM sodium acetate (pH 4.0) at 4° C.with several changes overnight.

[0063] An antibody solution of 10 mg/ml in 20 mM carbonate (pH 9.5) isprepared.

[0064] The HRP is removed from the dialysis tubing and added to 0.5 mlof the antibody solution and is incubated at room temperature for 2 hr.

[0065] The Schiff's bases, thus formed, are reduced by adding 100 ul ofsodium borohydride (4 mg/ml in water) and incubated at 4° C. for 2 hr.

[0066] The solution is dialyzed versus several changes of PBS.

[0067] iv) Antibody-enzyme conjugate (100 ul) prepared as in (iii) anddiluted 1:500 to 1:300,00 is added and reaction/readings taken as in[4a(iii)].

[0068] c) Competition ELISA

[0069] i) As for [4a(i)].

[0070] ii) Compounds/samples (100 ul) are added to wells andsimultaneously 100 ul antibody-enzyme conjugate is added [see 4b(iii+iv)]. The plate is incubated for 45 min at 37° C. and the proceduredescribed in [4a (iii)] carried out.

[0071] The results of this procedure are shown in FIG. 1.

RESULTS

[0072] Competition assays are performed to detect the free product in areal aqueous sample. OVA conjugate is bound to the walls ofmicrotitration wells and incubated with

[0073] 1) Polyclonal antiserum raised to the free form (dilution 1:100to 1:8000) and free product; range 10 ng/ml to 100 μg/ml (c.f. FIG. 1).

[0074] 2) Polyclonal antiserum raised to the KLH conjugate (dilution1:100 to 1:35,000) and free product range 10 ng/ml to 100 μg/ml.

[0075] 3) Monoclonal antibodies raised to the free form (dilution 1:10⁴to 1:10⁶) and free product range 10 ng/ml to 100 μg/ml

[0076] and

[0077] 4) Monoclonal antibodies raised to the KLH conjugate (dilution1:10⁴ to 1:10⁶) and free product range 10 ng/ml to 100 μg/ml.

[0078] Assays incorporating polyclonal or monoclonal antibodies to theconjugated form are sensitive only down to 10 μg/ml. Those incorporatingpolyclonal and monoclonal antibodies to the free form are sensitive downto 0.1 μg/ml (c.f. FIG. 1).

MATRIX INTERFERENCE

[0079] The product is prepared in a variety of synthetic waters and twoexamples of typical north sea formation water in which the product iscommonly applied, to determine matrix interference (see Table 1).

[0080] Absorbance (A450) of the positive polymer control in the presenceof distilled water is 1.68±0.19 AU.

[0081] A450 of the negative polymer control is 0.08±0.04 AU.

[0082] A450 in the presence of the synthetic waters and one of the northsea formation waters was >1.58±0.28 AU. The second formation waterbrought about a colour change when added to the tetramethylbenzidinesubstrate. TABLE 1 TYPE COMPOSITION FORMATION 1 Barium (Ba²⁺) 1050 ppmCalcium (Ca²⁺) 1060 ppm Magnesium (Mg²⁺) 113 ppm Sodium (Na⁺) 27,986 ppmChloride (Cl⁻) 43,196 ppm Potassium (K⁺) 3833 ppm Strontium (Sr²⁺) 110ppm SEAWATER 1 Sulphate (SO₄ ²⁻) 2426 ppm Sodium (Na²⁻) 22,135 ppmChloride (Cl⁻) 34,165 ppm Potassium (K⁺) 775 ppm Bicarbonate (HCO₃ ⁻)497 ppm THESE ARE MIXED 50/50 or 40/60 OF FORMA- TION 1/SEAWATER 1 andpH adjusted to 4.5 FORMATION 2 Barium (Ba²⁺) 252 ppm Calcium (Ca²⁺) 3523ppm Magnesium (Mg²⁺) 1813 ppm Sodium (Na⁺) 17,692 ppm Chloride (Cl⁻)39,599 ppm Strontium (Sr² ⁺) 669 ppm SEAWATER 2 Sulphate (SO₄ ²⁺) 2426ppm Sodium (Na²⁻) 22,135 ppm Chloride (Cl⁻) 34,165 ppm Potassium (K⁺)775 ppm Bicarbonate (HCO₃ ⁻) 497 ppm THESE ARE MIXED 50/50 OF FORMATION2/SEAWATER 2 FORMATION 3 Calcium (Ca²⁺) 467 ppm Magnesium (Mg²⁺) 75 ppmPotassium (K⁺) 377 ppm Strontium (Sr²⁺) 67 ppm Barium (Ba²⁺) 65 ppmSodium (Na⁺) 12,932 ppm Chloride (Cl⁻) 20,853 ppm SEAWATER 3 Bicarbonate(HCO₃ ⁻) 4000 ppm Sodium (Na⁺) 1,511 ppm THESE ARE MIXED 75/25 OFFORMATION 3/SEAWATER 3 SOLUTION 4 Calcium (Cl²⁺) 150 ppm Magnesium(Mg²⁺) 44 ppm Chloride (Cl⁻) 199 ppm Sodium (Na⁺) 121 ppm Carbonate (CO₃²⁻) 51ppm Bicarbonate (HCO₃ ⁻) 269 ppm SOLUTION 5 Calcium (Ca²⁺) 300 ppmMagnesium (Mg²⁺) 88 ppm Chloride (Cl⁻) 398 ppm Sodium (Na^(═)) 242 ppmCarbonate (CO₃ ²⁺) 102 ppm Bicarbonate (HCO₃ ⁻) 538 ppm SOLUTION 6Calcium (Ca²⁺) 20 ppm Magnesium(Mg²⁺) 6 ppm Chloride (Cl⁻) 30 ppmSulphate (SO⁴⁻) 21 ppm Bicarbonate (HCO₃ ⁻) 18 ppm Sodium (Na⁻) 46 ppmSOLUTION 7 Calcium (Ca²⁺) 60 ppm Magnesium (Mg²⁺) 18 ppm Chloride (Cl⁻)200 ppm Sulphate (SO⁴⁻) 200 ppm Bicarbonate (HCO₃ ⁻) 427 ppm Sodium(Na⁺) 83 ppm SOLUTION 8 Calcium (Ca²⁺) 400 ppm Magnesium (Mg²⁺) 1202 ppmChloride (Cl⁻) 18711 ppm Sodium (Na⁺) 10522 ppm Carbonate (CO₃ ²⁻) 184ppm Sulphate (SO₄ ²⁻) 2623 ppm Potassium (K⁺) 395 SOLUTION 9 Calcium(Ca²⁺) 172 ppm Sodium (Na⁺) 304 ppm Carbonate (CO₃ ²⁺) 153 ppmBicarbonate (HCO₃ ⁻) 129 ppm Chloride (Cl⁻) 400 ppm Sulphate (SO₄ ²⁻)159 ppm SOLUTION 10 Calcium (Ca²⁺) 100 ppm Magnesium (Mg²⁺) 20 ppmChloride (Cl⁻) 118 ppm Sodium (Na⁺) 50046 Hydroxide (OH⁻) 36167Carbonate (CO₃ ²⁻) 10556 Potassium (K⁺) 2422 SOLUTION 11 Calcium (Ca²⁺)23 ppm Magnesium (Mg²⁺) 10 ppm Silica (SiO₂) 28 ppm Carbonate (CO₃ ²⁺)226 ppm Phosphate (PO₄ ²⁻) 74 ppm Iron (Fe³⁺) 34 ppm SOLUTION 12 Typicalnatural sea Water sample SOLUTION 13 Typical north sea formation waterExample 1 SOLUTION 14 Typical north sea formation water Example 2

EXAMPLES 2 TO 26

[0083] The following compounds of similar structure are substituted inthe competion assay, in place of the free product, in the proceduredescribed in Example 1. The results as shown in Table 2 are expressed asa percentage ratio of the mass of polymer giving 50% maximum absorbanceto mass of compound of similar structure. The antibody is specific forthe determination of phosphinocarboxylic acids. TABLE 2 PERCENT CROSSREACTIVITY EX- WITH AMPLE COMPOUND TELOMER 1 2 ACRYLIC/ACRYLAMIDE METHYL2.6 PROPANOSULPHONIC ACID (AMPS) COPOLYMER 1 3 PHOSPHONIC CARBOXYLICACID PCA 105 4 PHOSPHONO CARBOXYLIC ACID 12.3 5 ACRYLIC/AMPS COPOLYMER 29.7 6 POLYACRYLIC ACID 1 5.4 7 POLYACRYLIC ACID 2 8.8 8 POLYACRYLIC ACID3 10.5 9 ACRYLIC COPOLYMER 22.7 10 POLYACRYLIC ACID 4 14.4 1ACRYLIC/AMPS COPOLYMER 3 12.7 12 ACRYLIC/AMPS/ 9.6 POLYETHYLENEGLYCOLCOPOLYMER 13 POLYACRYLIC ACID 5 6.6 14 1-HYDROXY ETHYLIDENE-1-1- 11.4DIPHOSPHINIC ACID (HEDP)/AMPS COPOLYMER/POLYACRYLIC ACID 15 PHOSPHONOBUTANE TRICARBOXYLIC 8.8 ACID (PBTC) 16 HEDP 2.1 17 PHOSPHONATE 1 2.6 18PCA 2 91.5 19 POLYACRYLIC ACID 6 3.0 20 PHOSPHONATE 2 8.4 21 PHOSPHONATE3 9.7 22 AMINE OXIDE OF AMINE PHOSPHONATE 1 15.6 23 AMINE OXIDE OF AMINEPHOSPHONATE 2 11.0 24 HYDROXYPHOSPHINOUS CARBOXYLIC 28.2 ACID 25ACRYLIC/AMPS COPOLYMER 4 11.5 26 ACRYLIC/AMPS COPOLYMER 5 7.7

EXAMPLE 27

[0084] Attempts to conjugate the telomer derived from 3 moles of maleicacid 1 mole of vinyl acetate and 1 mole of ethylacrylate with KLHresulted in total precipitation at all reasonable ratios of reactants,as described in Example 1. Low ratio coupling of the product to OVA withEDC is successful (1:4, by weight). In addition low ratio coupling to asecond protein, bovine serum albumin (BSA) for screening purposes isalso prepared.

[0085] Mice and rabbits are immunised as described in Example 1.Antibody production is determined after immobilisation of the secondBSA-conjugate onto the walls of a microtitration well and the proceduredescribed in Example 1 is performed.

[0086] The conjugated form of the product is shown to be immunogenic. Noresponse is detected from the free form. This is consistent with thesize of the molecule being too small (m_(w)<1000 daltons) to stimulatethe immune system.

1. A method for determining the presence and/or concentration of a watertreatment polymer in an aqueous sample, comprising producing apolyclonal or monoclonal antibody to the water treatment polymer, andusing the antibody so produced as a reagent in an immunoassay, conductedon the aqueous sample.
 2. A method according to claim 1 in which thewater treatment polymer has the formula I:

or salts thereof, in which R″ is hydrogen, methyl or ethyl, R ishydrogen, C₁-C₁₈ alkyl, C₅-C₁₂ cycloalkyl, aryl, aralkyl, a residue offormula:

in which R″ has its previous significance and the sum of m and n is aninteger of at most 100, or R is a residue —OX in which X is hydrogen orC₁-C₄ alkyl, and R′ is a residue —OX in which X has its previoussignificance.
 3. A method according to claim 2 in which the watertreatment polymer has the formula IA:

in which the sum of m′ and n′ is an integer ranging from 8 to
 32. 4. Amethod according to claim 3 in which the sum of m′ and n′ is
 16. 5. Amethod according to claim 1 in which the water treatment polymer is ahydrolyzed terpolymer of maleic anhydride with other monomers, the molarratio of maleic anhydride to other monomers ranging from 2.5:1 to 100:1,and the molecular weight of the terpolymer being below
 1000. 6. A methodaccording to claim 5 in which the other monomers are vinyl acetate andethyl acrylate, and ratio of maleic anhydride to the other comonomersranges from 2½-3½:1 by weight.
 7. A method according to claim 6 in whichthe ratio of maleic anhydride, to the other comonomers is 3:1 by weight.8. A method according to claim 1 in which the polymer is a polyacrylicacid polymer, a phosphinocarboxylic acid polymer, a copolymer of acrylicacid and acrylamidomethylpropane sulphonic acid (AMPS); copolymer ofacrylic acid and vinyl acetate; polymaleic acid; hydrolysed polymaleicacid; a terpolymer of maleic acid, ethyl acrylate and vinyl acetate; acopolymer of acrylic acid and maleic anhydride; a copolymer of maleicacid and sodium allyl sulphonate; a copolymer of maleic anhydride andsulphonated styrene; or a vinyl sulphonic acid telomer.
 9. A methodaccording to any of the preceding claims in which the aqueous sample istaken from that employed in cooling water plant steam generating plant,sea-water evaporators, reverse osmosis equipment, paper manufacturingequipment, sugar evaporator equipment, soil irrigation plant,hydrostatic cookers, gas scrubbing systems, closed circuit heatingsystems, aqueous-based refrigeration systems and down-well systems. 10.A hybridoma having the characteristics of ATCC or nutrients or variantsthereof.
 11. A composition for determining the presence and/orconcentration of a water treatment polymer in an aqueous sample,comprising an effective amount of a monoclonal or polyclonal antibodywhich has been produced from the polymer, in an acceptable carrier.